Sequence-selective DNA detection has become increasingly important as scientists unravel the genetic basis of disease and use this new information to improve medical diagnosis and treatment. DNA hybridization tests on oligonucleotide-modified substrates are commonly used to detect the presence of specific DNA sequences in solution. The developing promise of combinatorial DNA arrays for probing genetic information illustrates the importance of these heterogeneous sequence assays to future science.
Typically, the samples are placed on or in a substrate material that facilitates the hybridization test. These substrate materials can be glass or polymer microscope slides or glass or polymer microtiter plates. One example of a probe includes capture probes, such as DNA capture probes. Organization of the tests on a substrate may occur by laying out areas of circular patterns of concentrated capture strand DNA in nominal sizes between 100 and 500 microns. As shown in FIG. 1, there are 10 areas on the substrate. More or less areas may be used depending on the needs of experiments. Further organization may occur by placing spots with different synthetic DNA sequences in a common area that is exposed to the same sample. In particular, there may be a plurality of the same or different types or probes in an area on the substrate.
The DNA hybridization test may thus include: synthetic DNA capture strands immobilized on a substrate; a strand of target DNA; and a probe. Specifically, one such technique for DNA hybridization is the chip based DNA detection method that employs probes. A probe may use synthetic strands of DNA complementary to specific targets. Attached to the synthetic strands of DNA is a signal mechanism. If the signal is present (i.e., there is a presence of the signal mechanism), then the synthetic strand has bound to DNA in the sample so that one may conclude that the target DNA is in the sample. Likewise, the absence of the signal results (i.e., there is no presence of the signal mechanism) indicates that no target DNA is present in the sample. Thus, a system is needed to reliably detect the signal and accurately report the results.
One example of a signal mechanism is a gold nanoparticle probe with a relatively small diameter (10 to 40 nm), modified with oligonucleotides, to indicate the presence of a particular DNA sequence hybridized on a substrate in a three component sandwich assay format. See U.S. Pat. No. 6,361,944 entitled “Nanoparticles having oligonucleotides attached thereto and uses therefore,” herein incorporated by reference in its entirety; see also T. A. Taton, C. A. Mirkin, R. L. Letsinger, Science, 289, 1757 (2000). The selectivity of these hybridized nanoparticle probes for complementary over mismatched DNA sequences was intrinsically higher than that of fluorophore-labeled probes due to the uniquely sharp dissociation (or “melting”) of the nanoparticles from the surface of the array. In addition, enlarging the array-bound nanoparticles by gold-promoted reduction of silver(I) permitted the arrays to be imaged in black-and-white by a flatbed scanner with greater sensitivity than typically observed by confocal fluorescent imaging of fluorescently labeled gene chips. The scanometric method was successfully applied to DNA mismatch identification.
To execute the DNA hybridization, the user should locate together complementary strands of synthetic DNA with the target DNA at a specified temperature and humidity. The temperature should be closely controlled so that only the DNA of choice hybridizes, which increases the test's selectivity. Controlling the humidity is thus important as the fluid volumes used in the test are in the microliters range.
In order to process the test, the user should interact several reagents at very small volumes. Micropipettes may be used to transfer reagents from their storage containers into mixing containers. The mixing container is much larger than the fluid volumes used so a centrifugation step is necessary to condense all the solution into one area of the container. This mixing container must also be humidity and temperature controlled so it must be a closed environment that can be immersed in or placed on a medium that is maintained at the desirable hybridization temperature. One may use microfuge tubes, racks, an environmental chamber, water baths, vortexing machines and mini-centrifuges to execute this process.
In the prior art, the hybridized target DNA/signal mechanism (such as gold nanoparticle DNA) is added to a slide using a micropipette to transfer the solution from the mixing container to the slide. In this prior art method, a gasket is manually applied to the microscope slide using adhesive. A second hybridization step now occurs with the solution on the slide inserted into an environmental chamber to maintain the slides temperature and humidity. The slide is removed from the environmental chamber following the second hybridization and the excess fluid/unbound DNA is removed by washing the slide in a water-based wash solution.
The last step may be the addition of a signal amplification solution, which may precipitate a metal onto the signal mechanism. This process should occur with a controlled temperature, humidity and light conditions as the solution is very reactive to light and temperature. Once this step is complete, the metal precipitate solution is removed from the slide by a second water-based wash solution.
These steps used in the prior art are complex, but the process can be manually controlled when only a single sample is being tested. However, a typical scenario is for many different samples to be run through the process in parallel. This results in high amounts of complexity as many tubes laid out in rack systems must all be tracked by the user as they sequentially remove the correct volumes of solutions from each tube and placed it in another corresponding tube or in a specific area of the hybridization slide. It is common for mistakes in micropipetting, spatial mapping or task sequencing to render a DNA hybridization test useless. The prior art manual process is also difficult to control thermally.
Accordingly, it would be advantageous to have a device and a method that would allow a simplification of the above process.